Characterization of RNA Genetic Regulators and Synthetic Networks
Westbrook, Alexandra Michon
A central tenent of synthetic biology is the ability to predictably engineer complex patterns of gene expression. This fined tuned control allows us to reprogram organisms with sophisticated synthetic behaviors such as producing vital chemicals and drugs and sensing environmental signals. In order to do this we need libraries of highly efficient genetic regulators and proven methods of combining them into networks. RNA presents the ideal tool to build new genetic networks because its structural and temporal characteristics allow engineers to construct fast, designable genetic networks. In this work, we show characterization and optimization of new and existing RNA regulators as well as efforts to create new behaviors with RNA-based genetic networks. We begin by vastly improving the dynamic range of an existing transcriptional RNA regulator, the pT181 attenuator, by adding translational regulation. This dual control attenuator is successfully used to reduce circuit leak in an RNA-only cascade. In order to expand upon the functionality of the RNA repressors, we design sequesters that allow us to dial down repression. The sequestration effectively creates a threshold which we use to tune the relationship between the input and output of a system. As we construct more complex circuits with diverse parts, modularity becomes essential in order to predict circuit behavior. We explore the modularity of our RNA regulators in combination with clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) in construction of an RNA pulse generator. Finally, we explore the design and implementation two complex circuits: a communication network for delivering complex signals to cells and a control network to reduce noise in biological systems. We anticipate that the design rules learned and the tools developed here will allow construction of even more sophisticated behaviors as the growing discipline of genetic design matures.
RNA; Biological control; Bioengineering; Biological Networks; synthetic biology
Stroock, Abraham Duncan; Daniel, Susan
Ph. D., Chemical Engineering
Doctor of Philosophy
dissertation or thesis